ICAM-1 binding antibodies

ABSTRACT

The invention provides binding molecules, including antibody molecules which selectively bind to a cell surface antigen of a target cell, and wherein the binding molecules, on binding the cell surface antigen, induce apoptosis of the target cell. There is also provided methods of and pharmaceutical compositions for apoptosis induction and uses thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage application under 35 U.S.C. §371and claims benefit under 35 U.S.C. §119(a) of International ApplicationNo. PCT/EP2006/012065 having an International Filing Date of Dec. 8,2006, which claims the benefit of priority of GB 0525214.3 having afiling date of Dec. 12, 2005, all of which are incorporated herein intheir entirety.

The invention relates to molecules involved in apoptosis induction,methods and pharmaceutical compositions for apoptosis induction and usesthereof.

Antibodies have recently become the protein therapeutics of choice fortargeting cancer but also for treating other indications (Brekke et al.Nat Rev Drug Discov 2003; 2:52-62). The advent of antibody engineeringhas provided the tools to generate human antibodies from synthetic phagelibraries, displaying decreased immunogenicity and enhanced specificityand affinity due to their human nature and greater diversity (Weiner etal. Nat Biotechnol 2005; 23:556-7). Naïve libraries are particularlyattractive, as they may be used for isolation of antibodies for anyspecificity, including self-antigens (Griffiths et al. Embo J 1993;12:725-34), independent of immunizations and reconstruction of newlibraries. Cell surface receptors constitute by far the most successfulgroup of antigens targeted by contemporary therapeutic drugs, includingsmall molecule inhibitors and antibodies. Of particular interest arecell surface receptors that are uniquely expressed or that display anincreased expression level on a target cell and are additionally capableof relaying death or survival signals to the cell. Such differentiallyexpressed receptors with intrinsic signalling properties enableantibody-based targeting of microbial infected, transformed, orotherwise malfunctioning cells.

For treatment of tumours, antibodies that have the ability to induceapoptosis in a target tumour cell whilst sparing normal tissue are ofparticular interest. Several such antibodies are in use, have beenregistered with the US Food and Drug Administration (FDA) and providealternatives to conventional cancer treatments e.g. for lymphoma(rituximab targeting CD20) or for breast cancer (trastuzumab orcetuximab targeting Her-2 and EGFR respectively).

There are also other antibodies with apoptosis inducing effectscurrently in clinical development. However, even if these antibodiesdemonstrate beneficial effects in patients or in animal tests an unmetclinical need still exists.

Anti-idiotypic immunoglobulin targeting of B cell tumours was the firstmonoclonal antibody therapy conducted in man (Miller et al. N Engl J Med1982; 306:517-22.). Destruction of tumour cells by such means of passiveantibody administration (Riechmann et al. Nature 1988; 332:323-7.), oractive vaccination with the patients own tumour immunoglobulin protein(Kwak et al. N Engl J Med 1992; 327:1209-15.), has since beendemonstrated to confer tumour regression or tumour dormancy in patientswith different kinds of B cell malignancies. A more recent reportdescribes the generation of fully human anti-idiotype antibodies usingtransgenic mice deficient in mouse antibody production and expressingselected human antibody chain loci (Suarez et al. Mol Immunol 2004;41:519-26.).

In the present invention a competition biopanning method has been used,where target cell antigen in the form of whole cells, and excesssubtractor cell antigen in the form of membrane vesicles, are exposed atthe same time to the naïve n-CoDeR® antibody phage library (WO2004/023140; Soderlind et al. Nat Biotechnol 2000; 18:852-6.), toretrieve and subsequently test antibody fragments with excellentselectivity for B lymphoma target cells. Furthermore, functionality inthe selected binding molecules was demonstrated by the ability of theantibodies tested to induce apoptosis in target but not in non-targetcells.

Antibody specificities identified include HLA-DR/DP (the B1 antibody ofthe invention) and surface IgM (the C11 antibody of the invention), aswell as ICAM-1 (the B11 antibody of the invention), an adhesion moleculenot previously associated with apoptosis induction. Isolated antibodieshad affinities in the sub-nanomolar to nanomolar range, directly makingthem possible choices for targeted antibody therapy.

SUMMARY OF THE INVENTION

In a first aspect of the invention there is provided a method ofinducing apoptosis in a target cell comprising the steps:

-   a. providing one or more target cells displaying the cell surface    antigen, ICAM-1;-   b. providing one or more binding molecules which selectively binds    to cell surface ICAM-1 and, on binding ICAM-1, induces apoptosis of    the target cell;-   c. exposing the target cells of (a) to the binding molecules of (b)    to induce apoptosis in the target cells.

Preferably the binding molecule is an antibody molecule.

In a second aspect of the invention there is provided a method ofinducing apoptosis in a target cell comprising the steps:

-   a. providing one or more target cells displaying the cell surface    antigen, HLA-DR/DP and/or surface IgM;-   b. providing one or more antibody molecules which selectively binds    to cell surface HLA-DR/DP and/or surface IgM and, on binding    HLA-DR/DP and/or surface IgM, inducing apoptosis of the target cell;-   c. exposing the target cells of (a) to the antibody molecules of (b)    to induce apoptosis in the target cells.

In a third aspect of the invention there is provided a binding moleculewhich selectively binds to cell surface ICAM-1 and, on binding ICAM-1,induces apoptosis of the target cell. Alternatively the binding moleculeis an antibody molecule that selectively binds to cell surface HLA-DR/DPand/or surface IgM.

ICAM-I is also designated CD54, but for the purpose of this applicationICAM-1 will be used.

Binding molecules may be derived from antibodies and based on theantibody scaffold [Clackson T et al., Nature. 1991 Aug. 15;352(6336):624-8, Marks J D et al., J Mol. Biol. 1991 Dec. 5;222(3):581-97] that has been used extensively in many libraries, butbinding molecules may also be derived form other molecular scaffoldssuch as the fibronectin scaffold [Weng S et al., Proteomics. 2002January; 2(1):48-57] and the protein A scaffold [Nord K, et al., NatBiotechnol 1997 August; 15(8):772-7, Hogbom M et al., Proc Natl Acad SciUSA. 2003 Mar. 18; 100(6):3191-6]. Each of these scaffolds may havetheir advantages depending on application, and the antibody scaffold, asone example, may be used advantageously for creating variabilityindistinguishable from natural variability.

The basic structure of the antibody, the most commonly used scaffold, isvery well understood. In principle, a framework structure comprisingbeta strands ordered into two sheets present a set of variable loops,the so called Complementary Determining Regions (CDRs) that have thecapacity to bind to antigen molecules. Although antibodies may vary inthe scaffold structure the most extensive variability is seen in theCDRs. The great variability in-between antibodies, is the basis fortheir ability to interact, in a specific manner, with in principle alltypes of molecular structures. Due to this capacity, antibodies havebeen used extensively for generation of specific binders withapplicability within research, diagnosis/prognosis of disease and astherapeutic agents specific for defined target structures [Borrebaeck CA and Carlsson R, Curr Opin Pharmacol. 2001 August; 1(4):404-8].

Other, non-antibody binding molecules useful in this invention are thosehaving scaffold structures with a high degree of stability yet allowingvariability to be introduced at certain positions. An example of anotherbinding molecule is a fibronectin domain and a 58 amino acids largeprotein A domain which tolerate variability. There are also othermolecular folds that allow some degree of variation. Such examplesinclude major histocompatibility complex (MHC) class I and II moleculesand recently a novel class of molecules the so called defensins havebeen identified to be similar in basic structure while still harbouringextensive sequence variability in-between the gene family membersindicating that they are suitable as scaffolds for harbouring moleculardiversity. In addition, natural ligand(s) e.g. LFA-1 in the case ofICAM-1 as a target molecule, or recombinant variants of them, mayconstitute specific binding molecules able to induce apoptosis in targetcells.

Furthermore, the binding molecule may be any molecule selectivelybinding cell surface ICAM-1 of a target cell and, on binding, inducingapoptosis of the target cell.

The binding molecule is preferably an antibody molecule.

In one embodiment the cell surface antigen is ICAM-1.

The present screening retrieved an antibody (B11) specific for ICAM-1—areceptor not previously associated with apoptosis and not attributedintrinsic negative signalling properties in cells.

The identification of ICAM-1 as an apoptosis-inducing molecule was adirect result of the screening being designed to isolate specificitiesfor all surface receptors differentially expressed between target andnon-target cells, irrespective of and without prior knowledge of theirrespective identity. ICAM-1-induced cell death has been verified as anactive apoptotic process that involved mitochondrial membranedepolarization. Mitochondrial membrane depolarization has beenpreviously described for both caspase dependent and caspase independentapoptosis (Nagy et al. J Mol Med 2003; 81:757-65.).

The present findings further show that the epitope bound by the B11antibody is expressed in B lymphoma tissue of different origin, and isup regulated in certain B lymphoma cells compared to resting peripheralblood leukocytes. Importantly, in addition to B lymphoma cells alsocarcinoma cells expressing ICAM-1 underwent apoptosis when subjected tothe ICAM-1 specific B11 antibody in vitro (see Example 6).

Previous studies have demonstrated restricted expression of ICAM-1 onnormal human tissues (Smith et al. J Clin Pathol 1990; 43:893-900.).ICAM-1 is involved in cell to cell adhesion and plays an important rolein immune responses and inflammation through binding to its receptorLFA-1. Antibodies directed to ICAM-1 have been used to interfere withpathological immune responses and inflammation. In vivo administrationof a murine anti-ICAM-11 mAb in cymologous monkeys (Cosimi et al. JImmunol 1990; 144:4604-12.), or use in clinical trials in human patientswith rheumatoid arthritis or patients receiving kidney transplants hasalso revealed no overt toxicity (Kavanaugh et al. Arthritis Rheum 1994;37:992-9; Haug et al. Transplantation 1993; 55:766-72.).

The novel finding that ICAM-1 targeting can lead to apoptosisdemonstrates the possibility to use ICAM-1 specific binding molecules,such as antibodies for treatment of cancers of different originsprovided that they express the antigen.

Based on their expression of ICAM-1 cancer types that may potentially betreated with an apoptosis inducing anti-ICAM-1 antibody such as B11include: B lymphoma, myeloma (Huang et al. (1993) Hybridoma 12 p 661-75;Huang et al., (1995) Cancer Res 55 p 610-6; Smallshaw et al., (2004) JImmunother 27 p 419-24), gastric cancer (Maruo et al., (2002) Int JCancer 100 p 486-90), breast cancer (Rosette et al., (2005)Carcinogenesis 26 p 943-50), liver cancer (Sun et al., (1999) J CancerRes Clin Oncol 125 p 28-34), lung cancer (Grothey et al., (1998) Br JCancer 77 p 801-7), melanoma (Wang et al., (2005) Int J Cancer 27 p419-24), bladder cancer (Roche et al., (2003) Thromb Haemost 89 1089-97)and prostate cancer (Aalinkeel et al., (2004) Cancer Res 64 p 5311-21).Expression of ICAM-1 has also been identified in tumour metastasis asdemonstrated by (Maruo et al., 2002), (Rosette et al., 2005), (Sun etal., 1999), (Grothey et al., 1998), (Aalinkeel et al. 2004) pointing tothe possibility to intervene in metastasis processes using an ICAM-1specific antibody.

In a further embodiment the cell surface antigen is HLA-DR/DP.

HLA-DR/DP is normally present on, for example, B cells and can be foundup-regulated on B lymphoma cells.

To date, three different HLA-DR specific monoclonal antibodies haveentered clinical phase trials. The most recent addition of which is thefully human IgG₄ ID09C3, which was isolated from a similarly sized naïvephage library compared to n-CoDeR®, but using solid phase panning onpurified antigen (Nagy et al. Nat Med 2002; 8:801-7.).

In the present invention, a novel human antibody (B1) directed againstHLA-DR/DP that rapidly and with high potency induces apoptosis in amultitude of B-lymphoma cell lines has been identified therebydemonstrating HLA-DR/DP is linked to the induction of apoptosis in atarget cell.

The B1 antibody bound a multitude of B lymphoma cell lines of differentorigin (see Example 1 and Table 1) and was shown to induce apoptosis inHLA-DR/DP antigen expressing cells. Furthermore, the B1 antibody showeda higher potency than Rituximab when tested on the Raji B lymphoma cellline. This was particularly evident, when the IgG₄ format of the B1antibody was used (FIG. 8).

From the data obtained, this antibody has suitable characteristics fortreatment of HLA-DR/DP expressing B lymphoma cells. In addition, similarto the targeting of Rheumatoid Arthritis and SLE by Rituximab, the B1antibody may prove efficacious in eliminating activated B cells indisorders where HLA-DR/DP expressing B cells are detrimental.

In a yet further embodiment the cell surface antigen is surface IgM.

IgM in its free form exists as a large pentameric structure, whosehigher molecular weight tends to confine it within blood vessels.

Monomeric IgM can be found on the cell walls of B lymphocytes andfunctions as an antibody receptor for antigen recognition.

The C11 antibody of the invention, upon binding to surface IgM,expressed on B lymphoma cells, induces apoptosis in a rapid andefficient manner (see Example 1 and Table 1). In contrast to theidiotype specific anti-IgM antibodies previously used in the clinic fortreatment of B lymphoma the C11 antibody bind to a non-polymorphicepitope expressed on B cells from different donors and is thus suitablefor treatment of patients suffering from B lymphoma irrespective of Blymphoma idiotype.

Notably, the effector molecule e.g. the anti-IgM antibody could also beany type of specific binding molecule that causes apoptosis in IgMexpressing cells of different idiotypes.

The kinetics of B1, B11 and C11 IgG induced apoptosis were fast, withmaximal efficacy being observed already after 3 hours in some celllines. Rapid effector function is important for therapeutic efficacy asthis minimizes the risk for tumour evasion resulting from e.g. lack ofexpression of tumour antigen (Uyttenhove et al. J. Exp. Med. 1983;157:1040-52; Kennedy et al. Br J Haematol 2002; 119:412-6) or epitopemutation (Weiner et al. J Immunol 1989; 142:343-51; Bai et al. J. Clin.Invest. 2003; 111:1487-96), and potentially limits treatment durationand side-effects (Robert et al. Lancet Oncol 2005; 6:491-500.).

Preferably the target cell is an immune cell or epithelial cell andadvantageously that immune cell is a B lymphocyte.

Conveniently the target cell is associated with a disease. Preferablythe disease is selected from the group consisting of: cancer; autoimmunediseases including but not restricted to rheumatoid arthritis and SLE,acute and chronic inflammatory disorders, sepsis and infectious diseaseincluding but not restricted to HIV.

Advantageously, the disease is a cancer selected from lymphoma(leukaemia, myeloma), gastric cancer, breast cancer, liver cancer, lungcancer, melanoma, bladder cancer, chorioid cancer, pancreatic cancer,colon cancer and prostate cancer.

As defined in the definitions section of this application, the phraseantibody molecule is used for convenience and embraces, amongst otherthings, antibodies, an antibody fragments, and antibody derivatives.

Conveniently, the antibody molecule is an IgG. The IgG may be any ofIgG₁, IgG₂, IgG₃ or IgG₄, but preferably any of IgG₁ and IgG₄. Theantibody molecule is preferably humanized or human.

Conveniently, the binding molecule or antibody molecule of the inventionhas the sequence of any one of variable region sequences of FIGS. 9 to11 or functionally equivalent homologues thereof.

In one embodiment of the invention the binding molecule or antibodymolecule has the variable region sequences of FIG. 9 or functionallyequivalent homologues thereof.

In a further embodiment of the invention, the binding molecule orantibody molecule has the variable region sequences of FIG. 10 orfunctionally equivalent homologues thereof.

In a yet further embodiment of the invention, the binding molecule orantibody molecule has the variable region sequences of FIG. 11 orfunctionally equivalent homologues thereof.

In a fourth aspect of the invention there is provided a nucleic acidhaving a nucleotide sequence encoding a binding molecule or an antibodymolecule as claimed in any previous claim.

Conveniently the nucleic acid has the nucleotide sequence of any one ofFIGS. 9 to 11.

In a fifth aspect of the invention there is provided use of the bindingmolecule or antibody molecule as defined in the first or second aspectof the invention in the diagnosis and/or treatment and/or prevention ofa disease requiring the destruction of a target cell. There is alsoprovided the use of the binding molecule or antibody molecule as definedin the first or second aspect of the invention in the manufacture of amedicament for the treatment and/or prevention of a disease requiringthe destruction of a target cell.

In a preferred embodiment the binding molecule is an antibody molecule.

Conveniently, the disease to be treated is selected from the groupconsisting of: cancer; autoimmune diseases including but not restrictedto rheumatoid arthritis and SLE, acute and chronic inflammatorydisorders, sepsis and infectious disease including but not restricted toHIV.

Advantageously the disease to be treated is cancer selected fromlymphoma (leukaemia, myeloma), gastric cancer, breast cancer, livercancer, lung cancer, melanoma, bladder cancer, chorioid cancer,pancreatic cancer, colon cancer and prostate cancer.

In one embodiment of the invention the binding molecule or antibodymolecule binds specifically to ICAM-1 and/or has the sequence of FIG. 10and is used in relation to the diseases listed above.

In a further embodiment of the invention the antibody molecule bindsspecifically to HLA-DR/DP and/or has the sequence of FIG. 9 and is usedin relation to the diseases of lymphoma (leukaemia, myeloma), gastriccancer, breast cancer, liver cancer, lung cancer, melanoma, bladdercancer, chorioid cancer, pancreatic cancer, colon cancer and prostatecancer.

In a yet further embodiment of the invention the antibody molecule bindsspecifically to surface IgM and/or has the sequence of FIG. 11 and isused in relation to the diseases of lymphoma (leukaemia, myeloma),gastric cancer, breast cancer, liver cancer, lung cancer, melanoma,bladder cancer, chorioid cancer, pancreatic cancer, colon cancer andprostate cancer.

In a sixth aspect of the invention there is provided a pharmaceuticalcomposition comprising the binding molecule or antibody molecule of theinvention and a pharmaceutically acceptable carrier, excipient ordiluent.

In a preferred embodiment the binding molecule is an antibody molecule.

In a seventh aspect of the invention there is provided an in vitromethod of inducing apoptosis in a target cell comprising the steps of:

-   -   (i) providing one or more target cells;    -   (ii) providing one or more binding molecules or antibody        molecules as defined in the first embodiment of the invention;    -   (iii) exposing the target cells of (i) to the binding molecules        or antibody molecules of (ii) so as to induce apoptosis in the        target cells.

In a preferred embodiment the binding molecule is an antibody molecule.

Preferably the target cells provided in step (i) are immune cells orepithelial cells. Advantageously, the immune cells are B lymphocytes.

Conveniently, the target cells are associated with a disease and whereinthe disease is selected from the group consisting of: cancer; autoimmunediseases including but not restricted to rheumatoid arthritis and SLE,acute and chronic inflammatory disorders, sepsis and infectious diseaseincluding but not restricted to HIV.

Advantageously, the disease is a cancer selected from lymphoma(leukaemia, myeloma), gastric cancer, breast cancer, liver cancer, lungcancer, melanoma, bladder cancer, chorioid cancer, pancreatic cancer,colon cancer and prostate cancer.

Meanings of Terms Used

The term “antibody molecule” shall be taken to refer to any one of anantibody, an antibody fragment, or antibody derivative. It is intendedto embrace wildtype antibodies, synthetic antibodies, recombinantantibodies or antibody hybrids, such as, but not limited to, asingle-chain modified antibody molecule produced by phage-display ofimmunoglobulin light and/or heavy chain variable and/or constantregions, or other immunointeractive molecules capable of binding to anantigen in an immunoassay format that is known to those skilled in theart.

The term “antibody fragment” shall be taken to refer to any one of anantibody, an antibody fragment, or antibody derivative. It is intendedto embrace wildtype antibodies (i.e. a molecule comprising fourpolypeptide chains), synthetic antibodies, recombinant antibodies orantibody hybrids, such as, but not limited to, a single-chain modifiedantibody molecule produced by phage-display of immunoglobulin lightand/or heavy chain variable and/or constant regions, or otherimmunointeractive molecules capable of binding to an antigen in animmunoassay format that is known to those skilled in the art.

The term “antibody derivative” refers to any modified antibody moleculethat is capable of binding to an antigen in an immunoassay format thatis known to those skilled in the art, such as a fragment of an antibody(e.g. Fab or Fv fragment), or a modified antibody molecule that ismodified by the addition of one or more amino acids or other moleculesto facilitate coupling the antibodies to another peptide or polypeptide,to a large carrier protein or to a solid support (e.g. the amino acidstyrosine, lysine, glutamic acid, aspartic acid, cysteine and derivativesthereof, NH₂-acetyl groups or COOH-terminal amido groups, amongstothers).

The term “ScFv molecule” refers to any molecules wherein the V_(H) andV_(L) partner domains are linked via a flexible oligopeptide.

The terms “nucleotide sequence” or “nucleic acid” or “polynucleotide” or“oligonucleotide” are used interchangeably and refer to a heteropolymerof nucleotides or the sequence of these nucleotides. These phrases alsorefer to DNA or RNA of genomic or synthetic origin which may besingle-stranded or double-stranded and may represent the sense or theantisense strand, to peptide nucleic acid (PNA) or to any DNA-like orRNA-like material. In the sequences herein A is adenine, C is cytosine,T is thymine, G is guanine and N is A, C, G or T (U). It is contemplatedthat where the polynucleotide is RNA, the T (thymine) in the sequencesprovided herein is substituted with U (uracil). Generally, nucleic acidsegments provided by this invention may be assembled from fragments ofthe genome and short oligonucleotide linkers, or from a series ofoligonucleotides, or from individual nucleotides, to provide a syntheticnucleic acid which is capable of being expressed in a recombinanttranscriptional unit comprising regulatory elements derived from amicrobial or viral operon, or a eukaryotic gene.

The terms “polypeptide” or “peptide” or “amino acid sequence” refer toan oligopeptide, peptide, polypeptide or protein sequence or fragmentthereof and to naturally occurring or synthetic molecules. A polypeptide“fragment,” “portion,” or “segment” is a stretch of amino acid residuesof at least about 5 amino acids, preferably at least about 7 aminoacids, more preferably at least about 9 amino acids and most preferablyat least about 17 or more amino acids. To be active, any polypeptidemust have sufficient length to display biological and/or immunologicalactivity.

The terms “purified” or “substantially purified” as used herein denotesthat the indicated nucleic acid or polypeptide is present in thesubstantial absence of other biological macromolecules, e.g.,polynucleotides, proteins, and the like. In one embodiment, thepolynucleotide or polypeptide is purified such that it constitutes atleast 95% by weight, more preferably at least 99% by weight, of theindicated biological macromolecules present (but water, buffers, andother small molecules, especially molecules having a molecular weight ofless than 1000 daltons, can be present).

The term “isolated” as used herein refers to a nucleic acid orpolypeptide separated from at least one other component (e.g., nucleicacid or polypeptide) present with the nucleic acid or polypeptide in itsnatural source. In one embodiment, the nucleic acid or polypeptide isfound in the presence of (if anything) only a solvent, buffer, ion, orother component normally present in a solution of the same. The terms“isolated” and “purified” do not encompass nucleic acids or polypeptidespresent in their natural source.

The term “recombinant,” when used herein to refer to a polypeptide orprotein, means that a polypeptide or protein is derived from recombinant(e.g., microbial, insect, or mammalian) expression systems. “Microbial”refers to recombinant polypeptides or proteins made in bacterial orfungal (e.g., yeast) expression systems. As a product, “recombinantmicrobial” defines a polypeptide or protein essentially free of nativeendogenous substances and unaccompanied by associated nativeglycosylation. Polypeptides or proteins expressed in most bacterialcultures, e.g., Escherichia coli, will be free of glycosylationmodifications; polypeptides or proteins expressed in yeast will have aglycosylation pattern in general different from those expressed inmammalian cells.

The terms “selective binding” and “binding selectivity” indicates thatthe variable regions of the antibodies of the invention recognise andbind polypeptides of the invention exclusively (i.e., able todistinguish the polypeptide of the invention from other similarpolypeptides despite sequence identity, homology, or similarity found inthe family of polypeptides), but may also interact with other proteins(for example, Staphylococcus aureus protein A or other antibodies inELISA techniques) through interactions with sequences outside thevariable region of the antibodies, and in particular, in the constantregion of the molecule. Screening assays to determine bindingselectivity of an antibody of the invention are well known and routinelypracticed in the art. For a comprehensive discussion of such assays, seeHarlow et al. (Eds), Antibodies A Laboratory Manual; Cold Spring HarborLaboratory; Cold Spring Harbor, N.Y. (1988), Chapter 6. Antibodies thatrecognise and bind fragments of the polypeptides of the invention arealso contemplated, provided that the antibodies are first and foremostselective for, as defined above, full-length polypeptides of theinvention. As with antibodies that are selective for full lengthpolypeptides of the invention, antibodies of the invention thatrecognise fragments are those which can distinguish polypeptides fromthe same family of polypeptides despite inherent sequence identity,homology, or similarity found in the family of proteins.

The term “binding affinity” includes the meaning of the strength ofbinding between an antibody molecule and an antigen.

By the term “immune cell” we mean any cell that is involved in a hostimmune or inflammatory response, including but not limited to B cellsand T cells.

By the term “epithelial cell” we mean a cell of the epithelium.Epithelium is a tissue composed of a layer of cells. Epithelium can befound lining internal (e.g. endothelium, which lines the inside of bloodvessels) or external (e.g. skin) free surfaces of the body.

The outermost layer of our skin is composed of squamous epithelialcells, as are the mucous membranes lining the inside of mouths and bodycavities. Other epithelial cells line the insides of the lungs, thegastrointestinal tract, the reproductive and urinary tracts, and make upthe exocrine and endocrine glands. Functions of epithelial cells includesecretion, absorption and protection. Epithelial cells sit on a basallamina.

PREFERRED EMBODIMENTS

Examples embodying certain preferred aspects of the invention will nowbe described with reference to the following figures in which:—

FIG. 1—scFv Isolated by Differential Whole Cell/Cell Membrane VesicleBiopanning Show High Target Cell Specificity.

scFv clones isolated by differential biopanning were expressed in E.coli TOP10 cells and incubated with Ramos or Jurkat cells and (A) scFvclones expressed for primary screening or (B) seventy two randomlypicked and re-expressed scFv clones. Bound scFv was detected withanti-His MAb, and Cy5-anti-mouse polyclonal Ab. Cell binding wasdetected in an FMAT Macroconfocal High Throughput Screening instrument.Cell binding is depicted as mean fluorescence intensity to target Ramoscells (Y axis) vs. non-target Jurkat cells (X-axis). (C) Binding ofseven unique scFv clones to Ramos cells (filled bars) and Jurkat cells(open bars). A control scFv (ctrl) did not bind to any of the cells.

FIG. 2. Apoptosis Induction of Anti-Ramos scFv.

Ramos B lymphoma cells were sequentially incubated with anti-Ramos scFv,anti-His mAb, and anti-mouse polyclonal Ab on ice (with intermittentwashing to remove excess unbound antibody), and were incubated in ahumidified atmosphere of 5% CO₂ at 37° C. for 24 hours. Cells were thenharvested and subjected to combined staining with Annexin V-AF488 (AV)and propidium iodide (PI). Cells were scored as viable (AV− PI−, filledcircles FIG. 2C), early apoptotic (AV+ PI−, open triangles FIG. 2C), orlate apoptotic/necrotic (AV+ PI+, open diamonds FIG. 2C), based ondifferential positively for AV and PI staining (defined by square gatesin FIG. 2B). Results are presented by plotting (A) Forward Scatter(FSC-Height) against Side Scatter and (B) AV (FL-1) against PI (FL-3).The titratable effect of scFv B1 and F1 is also presented (C). The sevenunique scFv clones were incubated with (D) Ramos or (E) Raji B lymphomacells at 37° C. for 24 hours at various concentrations and the effect onapoptosis induction studied. Three scFv; B1, B11, and C11, showtitratable activity towards both cell lines, whereas apoptosis inducingcapability of scFv B10, C10, and G12 is restricted to Ramos B lymphomacells.

FIG. 3. Specificities of Isolated Antibodies Include HLA-DR/DP, IgM, andICAM-1.

A) 50-600×10⁶ Raji B lymphoma cells were lysed with the non-ionicdetergent Triton X-100 at 0.5% v/v and immunoprecipitated with 100 μg ofthe fully human IgG1 format of B1 (lane 1) and B11 (lane 2) antibody,followed by crosslinking with Protein A Sepharose. Ramos B lymphoma celllysates, from 50×10⁶ cells, were used for the precipitation of 20 μg C11(lane 3). Antibody-specific bands were excised and subjected to trypticdigestion and analysed by MALDI-TOF.

B) B1 IgG, B11 IgG, and C11 IgG binding to B lymphoma cells isspecifically blocked by pre-incubation with anti-HLA-DR/DP, anti-ICAM-1or anti-IgM antibodies, respectively.

To confirm the retrieved MALDI-TOF antigen identities of antibody clonesB1, B11, and C11, blocking studies with commercially availableantibodies was carried out and analyzed by flow cytometry. Cells werepre-blocked with 10-fold molar excess (compared to the human antibody)of species-matched blocking antibodies for 1 h, followed by the additionof any of the isolated human antibody clones. After 30 min, cells werewashed and binding of human antibody to cells was detected byPE-conjugated goat anti-human IgG (Caltag Laboratories, Burlingame,Calif., USA). The blocking antibodies used in the study were; for B1,mouse monoclonals anti-HLA DR (Sigma, clone HK14) or anti-CD40 (BecktonDickinson, clone 5C3); for B11, rabbit polyclonals anti-ICAM-1 (Abcam,ab7815-250) or anti-CD22 (Abcam, ab25135-100); for C11, goat polyclonalsanti-IgM (Zymed, South San Francisco, Calif., USA, 62-7500) or anti-IgG(Zymed, 62-8400).

FIG. 4. ICAM-1 is a B Lymphoma Associated Cell Surface Receptor Capableof Mediating Programmed Cell Death.

A. 2 μg/ml of B11 or anti-FITC-8 (control) IgG₁ was added to 4×10⁵ CL-01B lymphoma cells, incubated for 2 h on ice, followed by addition of 10μg/ml crosslinking secondary Fab′2 Goat anti-human Fc antibody. Cellswere incubated at 37° C. for 6 h and the effect of the antibodyincubation was determined by two independent apoptosis assays. Cellswere stained either by AV/PI (upper panel), similarly as describedabove, or by incubation with 5 μg/ml of the mitochondrial membranedepolarization reagent JC-1 for 30 min at RT (lower panel). Induction ofapoptosis is detectable by a decrease in the red (y-axis)/green (x-axis)fluorescence intensity ratio. (B) Histology section showingrepresentative binding of B11 antibody to B lymphoma tissue.Cryo-preserved tissue obtained from a patient with Anaplastic Large CellB Lymphoma was stained with B11 or FITC-8 (control) scFv antibody.Antibody binding was detected with DAB (brown colour). Inset pictureshows staining with control scFv. (C) CD45-PerCp-Cy5.5 mAb pre-labelledRamos cells were mixed with donor-derived PBMCs and the different cellpopulations were stained with fluorochrome-conjugated CD-specificantibodies and Alexa Flour 647 Zenon pre-labelled B11 IgG1 or controlFITC-8 IgG1. IgG B11 binding to the different cell populations wasrecorded in the FL4 channel.

FIG. 5. Affinities of IgG B1 and IgG B11 to B lymphoma cells.

Raji cells (left panels, IgG B1) or Ramos cells (right panels, IgG B11)were incubated with increasing amounts of radioiodinated IgG B1 orradioiodinated IgG B11 protein in the presence or absence of 0.2 mg/mlof the corresponding unlabeled IgG protein. Specific binding wasdetermined by subtracting binding in the presence of unlabeled competingprotein from total binding. The amount of bound IgG B1 or IgG B11protein increased with increasing amounts of free IgG protein withsaturable binding being reached at ˜30 nM IgG B1 and ˜1 nM IgG B11,respectively (upper panels). Rosenthal-Scatchard plot analysis (lowerpanels) demonstrated a dissociation constant of 3 nM with 400,000functional binding sites per Raji cell for IgG B1, and a dissociationconstant of ˜0.3 nM with 47,400 functional binding sites for IgG B11(Raji cells).

FIG. 6-Binding of B1, B11, C11 IgG₁ to tumour cell lines of differentorigin. The antigen distribution of antigens targeted by the B1, B11,and C11 antibodies on different carcinoma cell lines was investigated byflow-cytometry. Histograms show binding of Rituximab anti-CD20 Mab(first row front most peaks), B1 IgG₁ (second row peaks), B11 IgG₁(third row peaks), or C11 IgG₁ (fourth row back most peaks) to MCF-7breast carcinoma, HPAC pancreatic carcinoma, PC-3 prostate carcinoma,LS174 T colorectal carcinoma, and THP-1 monocytic leukaemia cells, asindicated.

FIG. 7—B11 IgG₁ Apoptosis Induction in Carcinoma Cells

The prostate carcinoma cell line PC-3 was grown in Complete GrowthMedium (RPMI 1640, supplemented with 10% FCS, 10 mM HEPES, and 2 mML-Glutamine) to 80% confluency in a 6 well plate. The prostate carcinomacell line DU145 was grown in MEM with Earl's salts, supplemented with10% FCS, 1 mM sodium pyruvate, and 1 mM non-essential amino acids andthe derivate of a melanoma cell line MDA MB 435 was grown in DMEMsupplemented with 10% FCS.

For apoptosis assays cells were washed in PBS and serially diluted B11IgG₁ (or B1 IgG₁, Trastuzumab or negative antibody control for controls)was added to individual wells and binding was allowed during a 1-2 hincubation at 4° C. The cells were washed and Complete Growth Medium wasadded, containing crosslinking antibody, Fab′2 Goat anti-Human Fab′2, at10 μg/ml. Cells were incubated in a humidified atmosphere, with 5% CO₂at 37° C., for 16-24 hours. Cells were collected by trypsination andstained with Alexa Fluor 488-Annexin V (AF488-AV) and propidium iodide(PI), according to manufacturer's instructions. The percentage apoptoticcells was determined by the formula: % apoptotic cells=100−%AF488−AV/PI−/−.

-   -   A) Contour plots show the relative distribution of PC-3 cells as        a function of Annexin V and Propidium Iodide positivity        following incubation as above with 2 μg/ml IgG B11 or IgG B1.    -   B) Bar graph shows the mean percentage of apoptotic PC-3 cells        following incubation with serially diluted B11 IgG₁ or 20 μg/ml        B1 IgG₁.    -   C) Bar graph shows the mean percentage of apoptotic MDA MB 435        cells following incubation with no antibody control, 10 μg/ml        negative antibody control, serially diluted B11 IgG₁, or 10        μg/ml Trastuzuimb IgG1.    -   D) Bar graph shows the mean percentage of apoptotic DU145 cells        following incubation with no antibody control, 10 μg/ml negative        antibody control, serially diluted B11 IgG₁, or 10 μg/ml        Trastuzumb IgG₁

FIG. 8. B1 IgG₁ and B1 IgG₄ Induce Direct Cell Cytotoxicity on Raji BLymphoma Cells in the Absence Of Cross-Linking Reagents.

Raji cells were incubated with B1 IgG₁, B1 IgG₄, Rituximab IgG₁, orcontrol CT-17 IgG₁ at 20, 6.7 or 2.2 μg/ml for 24 hours. Cells wereharvested and viability was determined as the percentage of Annexin Vand Propidium Iodide double negative cells.

FIG. 9—VH and VL sequences (nucleotide and amino acid sequences for B1antibody. SEQ ID NO:1 is the nucleotide sequence of B1-VH; SEQ ID NO:2is the amino acid sequence of B 1-VH; SEQ ID NO:3 is the nucleotidesequence of B1-VL; and SEQ ID NO:4 is the amino acid sequence of B 1-VL.

FIG. 10—VH and VL sequences (nucleotide and amino acid sequences for B11 antibody. SEQ ID NO:5 is the nucleotide sequence of B 11-VH; SEQ IDNO:6 is the amino acid sequence of B11-VH; SEQ ID NO:7 is the nucleotidesequence of B11-VL; and SEQ ID NO:8 is the amino acid sequence of B11-VL.

FIG. 11—VH and VL sequences (nucleotide and amino acid sequences for C11antibody. SEQ ID NO:9 is the nucleotide sequence of C11-VH; SEQ ID NO:10is the amino acid sequence of C11-VH; SEQ ID NO:11 is the nucleotidesequence of C11-VL; and SEQ ID NO:12 is the amino acid sequence ofC11-VL.

EXAMPLE 1 Selection and Screening (Biopanning) for Apoptosis InducingAntibodies with Specificity for B Lymphoma Associated Cell SurfaceReceptors

Cell Culture

The cell lines used in this study were obtained from ATCC (Manassas,Va., USA) or Deutsche Sammlung von Mikroorganismen und Zellkulturen(DSMZ) GmbH (Braunschweig, Germany) and were cultured in RPMI 1640medium supplemented with 10% FCS, 2 mM L-Glutamine, 10 mM HEPES and 1 mMNa pyruvate (all from Invitrogen, Carlsbad, Calif., USA) unlessotherwise stated. The Jurkat T leukaemia cell line (clone E6-1, TIB-152,ATCC), the B lymphoma cell lines DOHH-2 (ACC47, DSMZ), SC-1 (ACC558,DSMZ), WSU-NHL (ACC58, DSMZ), JVM-2 (ACC12, DSMZ), Jeko-1 (ACC553, DSMZgrown in 20% FCS), Rec-1 (ACC 584, DSMZ), SP-53 (Daibata et al. Cancer1989; 64:1248-53), RL (CRL-2261, ATCC), Granta 519 (DSMZ), NCEB-1(Saltman et al. Blood 1988; 72:2026-30), BJAB (Menezes et al.Biomedicine 1975; 22:276-84), Ramos (CRL-1596, ATCC), Raji (CCL-86,ATCC), Daudi (CCL-213, ATCC), CL-01 (Cerutti et al. J Immunol 1998;160:2145-57), the pre B cell lymphoma KM-3/Reh (CRL-8286, ATCC) and themultiple myeloma MC/CAR (CRL-8083, ATCC, grown in IMDM (Invitrogen)supplemented with 20% FCS) were all free of mycoplasma and cultured in ahumidified atmosphere at 37° C., using a 5% CO₂ atmosphere. The cellswere maintained at 2×10⁵−1×10⁶ cells/ml.

Jurkat Cell Membrane Vesicle Preparation

Jurkat cells were harvested by centrifugation at 300×g for 15 min in 500ml buckets (Corning Inc. Life Sciences, New York, USA), washed inDulbecco's PBS (Invitrogen), and resuspended in buffer A (1 mM NaHCO₃,1.5 mM MgAc, pH 7.4). Cell concentration was approximately 5×10⁷ Jurkatcells/ml (5×10⁹ cells in 100 ml Buffer A).

Cell disruption was achieved by hypo-osmotic shock treatment (Buffer A)on ice for 10-30 min and subsequent nitrogen cavitation in a Nitrogencavitation bomb (Parr Instrument Company, Moline, Ill., USA). Cells werekept at a constant pressure of 40 bar (4,000 kPa) for 15 min at 0° C.

Disrupted cells were collected in a 250 ml Sarstedt tube (Sarstedt AG &Co, Nümbrecht, Germany) containing 500 μl 0.5 M EDTA to yield a finalEDTA concentration of 2.5 mM. Addition of EDTA prevents aggregation ofmembrane vesicles. The homogenate (100 ml) was divided between 4×25 mlBeckman thick-walled rotor tubes (Beckman Coulter, Inc., Fullerton,Calif., USA), which were centrifuged for 10 min at 1900×g (4,000 rpm inan Sorvall SS34 rotor) at 4° C. to remove unbroken cells, nuclei, andheavy mitochondria.

The supernatant was collected and pelleted material was resuspended in25 ml of 1 mM NaHCO₃ buffer containing 1 mM EDTA and was re-centrifuged(further recovery of pelleted crude Jurkat membranes). Jurkat membraneswere pooled with membranes from the first centrifugation. Supernatantscontaining crude Jurkat membrane vesicles were ultra centrifuged using aBeckman Type 45Ti rotor at 40,000 rpm (approx. 200,000×g) for 2.5 h at4° C. Supernatants were poured off and remaining buffer was removed bytipping the tube edge against a tissue (e.g. Kleenex™).

The crude membrane pellet was transferred to a Dounce homogeniser withthe aid of a metal bar and was resuspended in 2.5 ml HES buffer (10 mMHepes, 1 mM EDTA, 0.25 M sucrose, pH 7.4) by several careful strokes inthe homogenizer. A membrane suspension concentration equivalent of 2×10⁹cells/ml containing 80-100 mg protein was, thus, achieved.

Selection of Phage Abs by Whole Cell/Cell Membrane Vesicle CompetitionBiopanning

Approximately 2×10¹³ phage particles were pre-warmed at 37° C. for 15min with intermittent mixing, centrifuged for 15 min at 14,000×g toremove precipitates, and the supernatant was transferred to a freshEppendorf tube. Non-fat dry milk was added to a final concentration of2% (w/v). Jurkat membrane vesicle preparations derived from 2×10⁹ cells(round 1 selection; 2×10⁸ cells round 2 and 3 selections) were thawed onice, and were mixed with the blocked phage particles. The mixture wasincubated for 15 min on ice.

5×10⁷ (5×10⁶ 2^(nd) and 3^(rd) rounds) Ramos cells were harvested bycentrifugation at 1,200 rpm for 6 min at 4° C. Supernatant was discardedand Ramos cells were resuspended in the milk-phage-Jurkat membranevesicle mixture. The suspension was incubated at 10° C. under slowend-over-end rotation for 4 h.

The cell/cell membrane vesicle/phage mixture was transferred to a 15 mlFalcon tube (BD Biosciences, Bedford, Mass., USA), containing 0.5 ml100% (trypan blue stained) Ficoll-Paque PLUS (Amersham Biosciences,Uppsala, Sweden) at the bottom, and 9.5 ml overlaid 40% (v/v) Ficoll in2% (w/v) BSA/PBS (Ficoll-pillar). The tube was centrifuged at 1,500 rpmfor 10 min at 4° C. The tube was removed from the centrifuge and thetube cap was screwed on and sealed airtight.

The bottom “tip” of the Falcon tube containing 100% Ficoll was choppedoff using a cigar-chopper. Thus, very high-density material includingmembrane vesicle sheets and cell nuclei were eliminated from the tube.The tube cap was then carefully opened disrupting the vacuum inside thetube and allowing liquid to be expelled drop-wise through the opening atthe (cut off) tube bottom.

The harvested cell suspension was washed once in PBS to remove excessFicoll. The pellet was resuspended in 1 ml PBS (not performed followingfinal wash) and the suspension was reloaded on top a fresh Ficoll-pillarand the washing procedure was repeated (twice in rounds 2 and 3).

Phage were eluted from cells by addition of 150 μl of 76 mM citric acid(pH 2.5) in PBS followed by incubation at room temperature for 5 min.The mixture was neutralized by addition of 200 μl of 1 M Tris-HCl, pH7.4. Supernatants containing eluted phage were saved following pelletingof cells at 300×g for 5 min. Further elution of phage was byresuspension and incubation of the cell pellet in 1 ml trypsin at RT for10 min.

Following inactivation with 40 μl 1 mg/ml aprotinin, cells werecentrifuged and supernatant containing eluted phage was saved. Elutedphage were used to infect Escherichia coli HB101F′ and the bacteria wereplated on TB medium containing appropriate antibiotics and glucose.Bacterial colonies were counted, scraped from the plates, and used asinoculums for the next round of panning.

Conversion to scFv Format, scFv, Expression, Purification, and Analysisof Cell Binding

The phagemid pool obtained following three rounds of selection wasdigested with EagI to remove the gene III. The resulting vector wasre-ligated. Vectors containing re-ligated uncut gene III fragments werelinearized by digestion with EcoRI enzyme. The scFv vector pool thusgenerated was used to transform E. coli TOP10 cells essentially asdescribed earlier (Soderlind et al. Nat Biotechnol 2000; 18:852-6.).

Bacteria were plated on large 500 cm² agar plates and individual cloneswere picked, transferred to 96-well plates, and expressed in TB mediumby over night culture at 37° C., 220 rpm using an automated system(Hallborn Biotechniques 2002; Suppl:30-7.). Recombinant scFv fragmentswere produced in TB medium containing appropriate antibiotics.

For primary screening of scFv clone binding to target Ramos cells andJurkat non-target cells, 5,000 Ramos or Jurkat cells were incubated witheither of 960 scFv clones, derived from the 3^(rd) round of selectionand produced as described above. Cells were incubated with 0.5 μg/mlanti-6×His mAb (R&D Systems, Minneapolis, Minn., USA) and 0.7 μg/mlCy5-conjugated Goat anti-mouse reagent (Amersham Biosciences). Cellbinding was analysed in an 8200 Cellular Detection System FluorescenceMacroconfocal High Throughput Screening (FMAT) instrument (AppliedBiosystems, Foster City, Calif., USA).

Following primary screening, seventy two bacterial clones were pickedrandomly (i.e. irrespective of target cell vs. non-target cellreactivity in the primary screening) for DNA sequencing as describedpreviously (Soderlind et al. Nat Biotechnol 2000; 18:852-6.) (Soderlindet al., 2000). For evaluation of cell surface binding by flow-cytometry,Ramos and Jurkat cells (both added at 2×10⁵ cells per test) wereincubated with individual scFv clones at a concentration of 2-10 μg/mlin PBS (Invitrogen) containing 0.5% w/v BSA (DPBS-B) for 1 h.

Cells were washed by centrifugation at 300×g for 6 min. Cells were thenincubated with FITC-conjugated CD19 mAb and PE-conjugated CD3 mAb (BD)enabling subsequent identification of target and non-target cells,respectively. Detection of scFv binding was achieved by incubation withRPE-Cy5-streptavidin (Dako Cytomation, Glostrup, Denmark) followingincubation with biotinylated anti-6×His mAb. Cells were incubated withsecondary and tertiary reagents for 40 min, and 15 min respectively. Allincubations were performed on ice using ice-cold solutions.

Differential Whole Cell/Cell Membrane Vesicle Panning

The present study utilized a novel panning protocol to isolateantibodies that target differentially expressed antigens in their nativecell surface configuration. Following three rounds of competitionbiopanning, using whole Ramos B lymphoma cells and membrane vesiclesderived from Jurkat T leukaemia cells, recombinant phage scFv wereisolated. These were converted to soluble scFv and expressed in E. coliTOP10 cells.

Recombinant scFv were incubated with target (Ramos) or non-target(Jurkat) whole cells and examined for cell binding. The specificity fortarget cell antigens of the antibody clones was striking, since 482 scFvclones expressed were shown to bind selectively to Ramos target cells atintensities ranging from weak to very strong (FIG. 1A). Only two cloneswere identified that weakly stained non-target Jurkat cells (FIG. 1A).

We next determined the genotype diversity of isolated phage displayedscFv. Seventy-two scFv clones were randomly picked (i.e. irrespective ofbinding tropism as determined in the primary screening) for DNAsequencing.

The clones were simultaneously re-expressed and re-evaluated for targetcell specificity (Ramos vs. Jurkat) by FMAT technology, as described(FIG. 1B). Seven different antibody genotypes were identified, asdetermined by their different CDRH3 and CDRL3 sequences (data notshown).

The high specificity of anti-Ramos scFv was confirmed by three colourflow-cytometric analysis, following incubation with equal numbers ofRamos and Jurkat cells and detection of scFv binding by means ofanti-tag antibody (FIG. 1C).

Target and non-target cells were defined by CD19 and CD3 expression,respectively, using fluorochrome conjugated CD specific monoclonalantibodies. The seven genotypically unique scFv clones showed high andvariable binding intensities to target Ramos cells, but no binding tothe non-target Jurkat cells, as compared to the negative control scFv.

Apoptosis Assay

Lipopolysaccharide levels of recombinantly produced scFv fragments werereduced using Detoxigel columns according to manufacturer's instructions(Pierce Biotechnology, Rockford, Ill., USA). Remaining endotoxin levelswere quantified by the LAL-amebocyte lysate assay (Cambrex Bioscience,Walkersville, Md., USA).

All scFv samples were found to contain less than 0.1 IU/ml oflipopolysaccharide. The chimeric anti-CD20 antibody Mabthera™(Rituximab) was purchased from Lund University Hospital (Lund, Sweden).2×10⁵ B lymphoma cells (Raji or Ramos) or Jurkat T cells were incubatedwith serially diluted and detoxified scFv's in culture medium for 1 h onice.

Cells were sequentially incubated with secondary anti-6×His mAb (5μg/ml), and tertiary Goat Fab′2 anti-mouse Fab′2 antibody (JacksonImmunoResearch Laboratories, Inc., West Grove, Pa., USA). Intermittentwashings ensured removal of excess unbound antibody reagent. Cells wereincubated at 37° C. in a humidified atmosphere of 5% CO₂ for 24 h.

When whole IgGs were used for apoptosis induction, crosslinking reagentwas replaced by goat Fab′2 anti-human Fc y antibody (JacksonImmunoResearch) with minimal cross-reactivity with non-IgG antibodyisotypes (to avoid unspecific crosslinking of endogenous B lymphomaassociated surface immunoglobulins) and incubated, as described above,for 6 h.

Apoptotic cells were, unless otherwise stated, detected by combinedstaining with Annexin V Alexa Fluor 488 (AV) and Propidium Iodide (PI)(both from Molecular Probes, Invitrogen) and subsequent flow-cytometricanalysis, Cells were defined as viable (AV−/PI−), early apoptotic(AV+/PI−) or late apoptotic/necrotic (AV+/PI+). AV and PI signals wererecorded in the FL1 and FL2 or FL3 channels (as indicated in the text),respectively, using a FACSCalibur instrument (BD Biosciences).

In order to investigate the functionality of the isolated scFv we set upa high throughput apoptosis screening assay, based on sequentialincubation and washing of cells with scFv and crosslinking reagent. Thedependence on scFv clone and concentration in the apoptosis assay isdemonstrated in FIG. 2 A-C, where the apoptotic effect of selected scFvclone B1 is compared to the—effect of scFv clone F1 which shows noinduction of apoptosis. Jurkat cells lacking target antigen expressiondid not die from apoptosis after treatment with any of the examined scFvdemonstrating that apoptosis induction depended on binding to targetantigen (data not shown).

Using the established scFv-apoptosis assay, we screened clones forapoptosis on Ramos and Raji B lymphoma cells. scFv-induced apoptoticeffects were compared to that induced by Rituximab anti-CD20 mAb (FIG.2D). Three scFv clones—B1, B11 and C11—were identified that inducedsignificant apoptosis on both Ramos and Raji cells (FIGS. 2 D and E).Induction of apoptosis by scFv on Raji cells correlated with binding tothese cells (FIG. 2D), since scFv clones that failed to bind Raji cellsdid not induce apoptosis.

The B1, B11 and C11 clones were transferred to fully human IgG1antibodies. Both their specificity and functionality remained intactafter reformatting, as demonstrated by their strong binding and potentcytotoxicity towards a broad panel of B lymphoma cell lines (Table 1).Notably, apoptosis induction was rapid with maximal percentage ofannexin V positive apoptotic cells being reached already after three tosix hours in several cell lines (Table 1 and data not shown).

IgG Production and Endotoxin Screening Assays

scFv antibody fragments were converted to full-length human IgG1λ formatvia cloning into a modified pcDNA3 vector (Norderhaug et al. J ImmunolMethods 1997; 204:77-87.), and transiently transfected into HEK293 cellsusing Lipofectamine 2000 reagent according to manufacturer'sinstructions (Invitrogen).

Human IgG was purified from spent cultivation medium on a MabSelectprotein A column (Amersham Biosciences). The purity of preparationswas >98% as determined by SDS-PAGE analysis. Antibody preparations werescreened and found to contain <0.1 IU/ml endotoxin at concentrationsused in the present study, and as determined by the LAL amoebocytelysate test (Cambrex Bioscience).

EXAMPLE 2 Analysis of Antibody Specificity

Antigen Identification

The identity of the targeted antigens was determined byimmunoprecipitation of B lymphoma cell lysates. Cells (50-600×10⁶ per mllysis buffer, depending on antibody and cell line) were harvested bycentrifugation, washed twice in PBS and incubated for 15 min in LysisBuffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, and CompleteEDTA-free Protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim,Germany)) containing the detergent Triton X-100 (Sigma-Aldrich, St.Louis, Mo., USA) at 0.5% v/v.

Cellular debris was spun down at 16,000×g for 15 min in a conventionaltable-top centrifuge and the soluble proteins were pre-cleared withProtein A Sepharose 4 Fast Flow (Amersham Biosciences) ( 1/10 volume ofreaction) for 1 h on rotation. For every sample, 1 ml of pre-clearedcell lysate was immunoprecipitated for 2 h by 20-100 μg of any of thehuman antibodies. Protein A Sepharose 4 Fast Flow was added again andincubated for 30 min, where after the immuno-complexes were washedextensively in lysis buffer, boiled for 5 min. and finally resuspendedin Sample Buffer (1× NuPAGE LDS Sample Buffer, 1× NuPAGE Sample ReducingAgent) and separated in a NuPAGE Novex 4-12% Bis-Tris Gel (all fromInvitrogen).

After staining (Simply Blue Safestain, Invitrogen), protein bands ofinterest were excised from the SDS-PAGE and subjected to trypticdigestion, as described (Edvardsson et al. Electrophoresis 1999;20:935-42.).

Briefly, gel plugs were destained and equilibrated by washing threetimes with 200 μl 50% acetonitril (ACN) under agitation. After drying ina SpeedVac concentrator (Savant, Farmingdale, N.Y., USA) for 15 min,samples were reduced by addition of 25 μl 10 mM DTT/100 mM NH₄HCO₃ andincubated for 56° C. for 1 h and alkylated by addition of 25 μl 55 mMiodoacetamide/100 mM NTH₄HCO₃ followed by incubation for 45 min at roomtemperature.

After two additional 10 min washing steps in 100 mM NH₄HCO₃ followed byone wash in 50% v/v ACN, the gel pieces were dried in a SpeedVacconcentrator and re-swelled and digested in 15 μl of 15 ng/μl trypsin(Promega Corporation, Madison, Wis., USA) in 25 mM NH₄HCO₃ at 37° C.over night. Peptides were extracted by addition of 50% v/v ACN/1% v/vTFA and 10 min incubation at RT. 1 μl of the extract was spotted ontoMALDI sample plates and allowed to dry. 1 μl matrix solution (5 mg/mlalpha-cyano-4-hydroxy cinnamic acid (CHCA) in 75% v/v ACN/1% v/v TFA)was spotted on top of the peptides.

Peptide masses were determined using an Applied Biosystems 4700 MaldiWorkstation. The proteins were identified by peptide mass fingerprintdatabase searching using Mascot search tools (Matrixscience, UK).Antigen specificities of clones B10, C10, and G12 were identified usingsimilar methodology, except scFv's and anti-His coated magneticmicrobeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) were usedfor immunoprecipitations.

Following conversion to the full antibody format B1, B11 and C11 IgGwere used to precipitate antigens from Raji and Ramos B lymphoma cells.IgG B1 precipitated two bands of approximately 28 and 34-kDa,respectively (FIG. 3A, lane 1). Gel slices containing these bands wereprepared and digested with trypsin and analysed by mass spectrometryidentifying HLADR/DP as the target antigen.

The specificity of IgG B1 for HLA-DR/DP was verified both by westernblotting and detection of HLA-DR/DP protein, using a commerciallyavailable monoclonal antibody, and by blocking of B1 IgG bindingfollowing pre-incubation of cells with a commercially available HLA-DRspecific monoclonal antibody (FIG. 3B).

The identities of the IgG B11 and C11 defined antigens were establishedusing similar methodology. IgG C11 was found to precipitate a 68 kDaprotein band identified as the membrane bound form of the B cellreceptor μ chain (FIG. 3A, lane 3). IgG11 precipitated a 90 kDa proteinband that was identified as the intercellular cell adhesion molecule-1(ICAM-1) (FIG. 3A, lane 2). The specificities of IgG B11 for ICAM-1, andof C11 IgG for IgM, were confirmed by MS-MS analysis, antibody blockingstudies (FIG. 3B), and western blot analysis (data not shown) usingcommercially available antibodies.

Specificities of clones B10, C10, and G12 were determined, using scFvand anti-H is coated magnetic microbeads for immunoprecipitation. Thethree scFv clones precipitated a protein band of 68 kDa, and MS-analysisof trypsin digested gel slices containing these bands revealed theirspecificity for surface IgM. Presumably, these antibodies recognize theRamos IgM idiotype, since neither of them cross-react with peripheralblood B lymphocytes or other IgM positive B cell lines.

EXAMPLE 3 Analysis of Antibody Affinities

In Vitro Iodination of B1 and B11 Immunoglobulins

Iodination of 1 mg/ml of IgG₁ B1 or IgG₁ B11 proteins with [¹²⁵I] NaIwas performed in PBS for 10 min using Iodogen pre-coated iodinationtubes (Pierce). Free [¹²⁵I] NaI was removed by desalting on PD-10columns (Amersham Biosciences), yielding specific radioactivities in therange of 100-1600 cpm per ng protein. [¹²⁵I] IgG₁ B1 and [¹²⁵I] IgG₁ B11was used for determination of antibody affinities.

Determination of IgG B1 and IgG B11 Affinity Constants

Radioiodinated IgG B1 or IgG B11 was incubated with B lymphoma cells inDPBS-B-hIgG (DPBS-B containing 0.2 mg/ml human IgG) for 2 h on ice withintermittent mixing. Non-specific binding was determined in the presenceof 0.2 mg/ml unlabeled IgG B1 or IgG B11 protein, as appropriate.Analysis was performed in triplicates.

Cells were loaded on top 40% v/v Ficoll/DPBS-B cushions in individualtubes and were centrifuged at 400×g for 6 min at 4° C. Samples werefrozen at −80° C. Cell pellets and cell supernatants were isolated andanalysed separately for ¹²⁵I-IgG protein content in a gamma counter,following cutting of the tubes in half.

Antibody affinity constants (Kd values) and epitope numbers per cellwere determined from Scatchard plot analysis according to Rosenthal etal (Anal Biochem 1967; 20:525-32), Bylund and Yamamura (Methods inNeurotransmitter Analysed. New York: Raven Press Ltd., 1990), andMarquardt (J. Soc. Indust. Appl. Math 1963; 11:431-41), as previouslydescribed (Brix et al. J. Clin. Invest. 1998; 102:283-93).

IgG B1 and IgG B11 binding to HLA-DR and ICAM-1 was characterised byincubating the radio-iodinated proteins with Raji or Ramos cells in thepresence or absence of 0.2 mg/ml of the corresponding unlabeled IgGprotein at 4° C. The specific binding of [¹²⁵I] IgG to the cell surfacewas calculated by subtracting non-specific binding (binding in thepresence of excess unlabeled IgG) from the total binding.

Saturation of specific IgG B1 binding to Raji cells was reached at ˜30nM IgG B1 (FIG. 5). Rosenthal-Scatchard plot analysis revealed adissociation constant of ˜3 nM with 400,000 functional binding sites perRaji cell, assuming a bivalent epitope-IgG interaction (FIG. 5).Similarly, the dissociation constant of IgG B11 was determined to ˜0.2nM with 47,400 receptors per Ramos cell.

TABLE 1 Fully human B1, B11 and C11 IgG antibodies show dynamic bindingpatterns and induce apoptosis in various B lymphoma cell lines. Antibodyclone -- specificity B1 - HLA Rituximab - DR/DP B11 - ICAM-1 C11 - IgMCD20 Tumor Apoptosis Apoptosis Apoptosis Apoptosis classification Cellline MFI^(b) Induction^(a) MFI Induction MFI Induction MFI InductionFollicular DOHH-2 140 − 100 − 90 − 480 ++ Lymphoma WSU- 280 + 0 − 60 −790 + NHL SC-1 170 + 0 − 50 − 50 − RL 50 − 100 − 210 − 200 + MantleGranta 370 ++ 260 + 60 + 360 +++ cell 519 Lymphoma JVM-2 650 + 100 − 10− 520 + Rec-1 0 − 380 − 900 − 580 + SP-53 500 ++ 90 − 360 − 740 ++NCEB-1 750 + 340 + 10 − 430 + Jeko-1 1000 +++ 30 + 1040 ++ 1160 +++Burkitt's Ramos 125 + 100 ++ 240 +++ 300 +++ Lymphoma Raji 550 +++ 420 +20 + 400 + Daudi 200 + 150 + 450 + 480 ++ BJAB 530 + 310 + 510 + 530 ++CL-01 940 +++ 600 ++ 60 + 970 ++ pre B cell Reh/KM-3 240 +++ 20 − 0 − 0− Leukaemia Multiple MC/CAR 290 ++ 120 + 0 − 110 − Myeloma ^(a)ApoptosisInduction; determined by percentage of viable cells after 6 hourincubation with any of the human antibodies, crosslinked with Goatanti-Human (gamma) Fc specific antibody; −, viability not affected; +,95-80%; ++, 79-60%; +++, 59-40% viable cells compared to control (humanFITC-8 IgG₁). The results are based on duplicate samples in threeindependent experiments. ^(b)MFI; Mean Fluorescence Intensity ofsecondary RPE-conjugated Goat anti-Human IgG antibody. The cell linedependent MFI value of control human FITC-8 IgG antibody was subtractedfrom the MFI of each human antibody.

EXAMPLE 4 ICAM-1 is a B Lymphoma Associated Antigen with ApoptosisInducing Properties

Flow-Cytometric Analysis of IgG Binding to Ramos Cells

Ramos cells (13×10⁶) were stained with CD45-PerCp-Cy5.5 mAb byincubation on ice for 45 min, washed in DPBS-B, and kept on ice untilmixing with unlabeled purified PBLs.

Buffy coats from two healthy volunteers were obtained from the LundUniversity Hospital. Buffy coats were diluted 1:2 in PBS and washed bycentrifugation at 500×g (1500 rpm Beckman Spinchron centrifuge) for 10min, complete aspiration of the supernatant and resuspension in DPBScontaining 1% heat inactivated FCS (DPBS-HI). Washing was repeatedtwice. Red blood cells were lysed by incubation with red blood celllysing solution (BD Biosciences) for 15 min at RT. Cells were washed bycentrifugation at 60×g (667 rpm Beckman spinchron centrifuge) for 10 minand the supernatant was carefully aspirated. Cells were counted in aBürker chamber following staining with trypan blue reagent (Invitrogen)and exclusion of dead cells, washed in DPBS-HI, pelleted, andresuspended in DPBS-B containing 200 μg/ml human purified IgG (blockingof Fc receptors).

For each donor and test condition, approximately 2.5×10⁶ leukocytes weremixed with 1.6×10⁵ PerCpCy5.5 pre-labelled Ramos cells. Mouse monoclonalCD3-FITC, CD56-PE, and CD19-PerCpCy5.5 antibodies (BD Biosciences) wereadded and the mixtures were incubated on ice until addition of labelledhuman IgG. Labelling of n-CoDeR human IgG antibodies and positivecontrol anti-CD20 mouse-human chimeric antibody Rituximab with AF647 Fabfragments (Molecular Probes, Invitrogen) was performed according tomanufacturer's instructions.

Briefly, 4 μg of each of IgG B1, B11, C11, and Rituximab antibodies wereincubated with 20 μl of AF647-Fab labelling reagent for 5 min at RT.Following addition of 20 μl human IgG blocking reagent and a furtherincubation for 5 min, AF647-labeled IgG was three-fold serially dilutedin DBPS-B, and diluted IgG proteins were added to the mixed Ramos/PBLcell solutions.

Samples were incubated for 1 h, washed, resuspended in DPBS-B, andanalysed for binding to different cell subpopulations by flow-cytometry,following appropriate calibration and compensation of the instrument forfour-colour analysis. Ramos cells were identified as thePerCpCy5.5^(high) population distinct from the B lymphocytePerCpCy5.5^(low) population.

Immunohistochemistry

Cryopreserved lymph node biopsies of patients with Anaplastic Large CellB lymphoma (one patient), Centroblastic/Centrocytic B non-Hodgkinlymphoma (three patients), and B cell chronic lymphocytic leukaemia (onepatient) were obtained from the Department of Pathology at LundUniversity (Lund, Sweden). Eight-micrometer sections of cryo-preservedtissue were fixed in acetone for 10 min at 4° C. Endogenousbiotin-binding activity was blocked by sequential treatment with Avidinand Biotin (Avidin/Biotin blocking kit, Invitrogen) for 20 min each.

Tissues were incubated with 5 μg/ml control scFv or B11 scFv for 1 h.Following washing, sections were incubated with biotin-conjugated mouseanti-His mAb (R&D Systems) for 30 min. scFv binding was detectedfollowing treatment with ABC Complex/HRP reagent (Dako Cytomation) for30 min, and subsequent incubation with DAB for 5 min.

Sections were photographed using a Leica DC 300F digital camera mountedon top of a Leica DMR light/fluorescence microscope.

Handling of human tissue followed the recommendation of the local EthicsCommittee at Lund University Hospital.

Mitochondrial Membrane Depolarization Assay

Mitochondrial membrane depolarization was analysed as previouslydescribed (Kim et al. Mol Biol Cell 2004; 15:420-34). Briefly,antibody-treated cells were mixed with JC-1 reagent (Molecular Probes)at 5 μg/ml and incubated for 30 min at RT. Cells were washed twice inice-cold PBS and resuspended in 300 μl PBS and analysed on a FACS Aria(BD Biosciences). The green and red fluorescence was collected through494/518 nm (FL-1) and 595/615 nm (FL-2) bandpass filters, respectively.

ICAM-1 is a glycoprotein of the immunoglobulin superfamily (Marlin etal. Cell 1987; 51:813-9) capable of inducing bi-directional signalling(Rothlein et al. J Immunol 1994; 152:2488-95; Vyth-Dreese et al. Blood1995; 85:2802-12). ICAM-1 has not previously been demonstrated to beinvolved in programmed death in B lymphoma cells.

Therefore, we wanted to confirm that IgG B11 induced cell death was anactive process, by means other than cell membrane phosphatidyl serinetranslocation.

Mitochondrial membrane depolarization was chosen as a validation ofapoptosis, since this is a common feature of caspase dependent andcaspase independent apoptosis that may be monitored by cell stainingwith5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanineiodide (JC-1 reagent).

In accordance with our Annexin V/propidium iodide assay (FIG. 4A, upperpanel), IgG B11 was found to induce mitochondrial membranedepolarization in CL-01 B lymphoma cells, as determined byflow-cytometric analysis following staining with JC-1 reagent (FIG. 4A,lower panel).

In order to exclude the possibility that ICAM-1 expression was an invitro artifact, resulting from a general up-regulation during cellculture, we examined the binding of IgG B11 to tissue obtained from fivedifferent patients with different B lymphoma tumours.

By immunohistochemistry, IgG B11 showed strong binding to the fivelymphoma tissues (FIG. 4B), at intensities comparable to, or slightlylower than, the anti-HLA-DR/DP antibody IgG B1 (Table 2).

We next examined the binding of IgG B11 to B lymphoma versus restingperipheral blood leukocytes. Ramos was chosen as a representative Blymphoma cell line, based on its low-end epitope expression yetsignificant sensitivity to B11 induced apoptosis. Flow-cytometricanalysis, following mixed incubation of pre-labelled Ramos cells withwhole blood peripheral blood leukocytes and either of IgG B1, B11 or C11antibodies, revealed that IgG B11 showed strong binding to Ramos cells.

Even more importantly, B11 demonstrated the greatest differentialbinding (strongest antigen up-regulation) of the three antibodies toRamos B lymphoma cells versus normal peripheral blood leukocytes (FIG.4C. and data not shown). IgG B11 binding peaked already at 0.1 μg/ml andwas 3.7-fold up regulated on Ramos versus monocyte cells (MFI 654 versus176), 8.3-fold up regulated on Ramos versus peripheral blood Blymphocytes (MFI 654 versus 78), and 23-fold up regulated compared to NKcells. Binding to other monitored peripheral blood leukocyte subsets wasnegative.

TABLE 2 ICAM-1 is strongly expressed in B lymphoma tissue of differentorigin Patient Antibody clone - specificity ID Tumour classificationB11-ICAM-1 B1-HLA-DR/DP A B-CLL^(c) (low malignant + ++ non-HodgkinLymphoma) B Anaplastic Large Cell ++ +++ B Cell Lymphoma CCentroblastic-Centrocytic ++/+++ ++ B non-Hodgkin Lymphoma DB-CLL/B-PLL^(d) ++ +++ E Centroblastic B ++/+++ +++ non-Hodgkin LymphomaF Centroblastic-Centrocytic ++ +++ B non-Hodgkin Lymphoma ^(c)B-CLL = B-Chronic Lymphocytic Leukemia, ^(d)B-PLL = B- Pro Lymphocytic Leukemia.Increasing numbers of + indicate stronger staining.

EXAMPLE 5 Antigen Distribution of B1, B11, C11 IgG₁ on Tumour Cell Linesof Various Origins, as Determined by Flow Cytometry

The antigen distribution of human antibody targeted antigens, mainly forB11, on different carcinoma cell lines was investigated. Cells (MCF-7and MDA MB 435S breast carcinoma, JAR and JEG-3 chorio-carcinoma, A549lung carcinoma, TCC-SUP urinary bladder carcinoma, MDA MB 435 melanoma,HPAC, PANC-1 and BxPC-3 pancreatic carcinoma, PC-3 and DU145 prostatecarcinoma, LS174 T, CaCO₂, and Lovo colorectal carcinoma, and THP-1monocytic leukaemia cells), were washed in PBS, and resuspended at 4×10⁶cells/ml in Complete Medium (200,000 cells/50 μl sample). B1 IgG₁, B11IgG₁, C11 IgG₁, negative control FITC-8 IgG₁, and Rituximab anti-CD20mAb was 3-10-fold serially diluted (10-0.1 pg/ml) in Complete Medium (50μl/sample). Cells were incubated with either of the antibodies for 1hour on ice, washed by resuspension in PBS/BSA 0.5%, centrifuged at 1200rpm for 5 min, and complete aspiration of the supernatants wasundertaken. Cells were incubated with PE-conjugated Goat F(ab′)2anti-Human IgG (Caltag Laboratories, Cat no: H10104), diluted 1/50 inPBS/BSA 0.5%, for 30 min, on ice. Following resuspension of in 300 μlPBS/BSA 0.5%, cells were analysed for IgG binding using a FACScaninstrument.

PC-3 prostate carcinoma cells showed strong expression of ICAM-1 asdemonstrated by the strong binding of B11 IgG to these cells (FIG. 6).MCF-7 breast carcinoma, HPAC pancreatic carcinoma, and LS174 Tcolorectal carcinoma cells were also found to express ICAM-1 albeit atlower intensity compared to the prostate cancer cells. In contrast,THP-1 monocytic leukaemia cells did not express ICAM-1. All carcinomacell lines initially tested were found negative for CD20, HLA-DR/DP, andIgM expression as demonstrated by the lack of binding of Rituximab IgG,B1 IgG, and C11 IgG, respectively. Further studies on additionalcarcinoma cells lines indicated that all carcinoma cells examined werepositive for ICAM-1 expression (Table 3).

TABLE 3 ICAM-1 is strongly expressed in carcinoma cell lines ofdifferent origin Tumor cell type Cell line MFI Chorio-carcinoma JAR 2000JEG-3 1600 Prostate carcinoma DU145 2200 PC-3 1500 Pancreatic carcinomaBxPC-3 2000 PANC-1 3800 Colon carcinoma CaCo2 800 Lovo 1600 Lungcarcinoma A549 800 Urinary bladder carcinoma TCC-SUP 3200 Melanoma MDAMB 435 4000 Mammary carcinoma MDA MB 435S 800

EXAMPLE 6 B11 IgG₁ Apoptosis Induction in Carcinoma Cells

B11 IgG₁ was shown in example 5 to bind strongly to carcinoma cells. Thepresent example examined the apoptosis inducing properties of thisantibody on carcinoma cells.

Cells were seeded in 6 well plates with Complete Growth Medium threedays before the onset of the experiment. Cells were between 50-75%confluent at the time of the experiment. Cells were washed with ice-coldPBS and incubated with serially diluted (20-0.02 μg/ml as indicated inthe figures, in 1 ml Complete Growth Medium) B11 IgG₁, 20 μg/ml controlB1 IgG₁, 10 μg/ml negative control IgG₁ or 10 μg/ml Trastuzumab IgG₁ asindicated at 4° C. for 1-2 hours. Cells were washed with ice-cold PBSand secondary F(ab′2) Goat anti-Human F(ab′2) antibody (diluted inComplete Growth Medium to 10 μg/ml) was added. Cells were incubated at37° C., in a humidified atmosphere of 5% CO₂ for 16-24 hours. Totalcells were collected by first isolating the supernatant, followed by PBSwash and trypsination of remaining adherent cells. The enzymaticreaction was terminated by resuspension in PBS containing 10%heat-inactivated fetal calf serum. Cells were washed in ice-cold PBS,subjected to Annexin V/propidium iodide staining, and analysed forviability/apoptosis as described in example 5 above.

The B11 IgG₁ was shown to induce apoptosis in the carcinoma cell linesin a specific and titratable manner (FIG. 7). Control IgG B1, which didnot bind to PC-3 cells (see example 5), also did not induce apoptosis inPC-3 cells. Negative control IgG₁ or Trastuzumab IgG₁ were not able toinduce apoptosis in DU145 or MDA MB435 cells.

EXAMPLE 7 Pharmaceutical Formulations and Administration

A further aspect of the invention provides a pharmaceutical formulationcomprising a compound according to the first aspect of the invention inadmixture with a pharmaceutically or veterinarily acceptable adjuvant,diluent or carrier.

Preferably, the formulation is a unit dosage containing a daily dose orunit, daily sub-dose or an appropriate fraction thereof, of the activeingredient.

The compounds of the invention will normally be administered orally orby any parenteral route, in the form of a pharmaceutical formulationcomprising the active ingredient, optionally in the form of a non-toxicorganic, or inorganic, acid, or base, addition salt, in apharmaceutically acceptable dosage form. Depending upon the disorder andpatient to be treated, as well as the route of administration, thecompositions may be administered at varying doses.

In human therapy, the compounds of the invention can be administeredalone but will generally be administered in admixture with a suitablepharmaceutical excipient, diluent or carrier selected with regard to theintended route of administration and standard pharmaceutical practice.

For example, the compounds of the invention can be administered orally,buccally or sublingually in the form of tablets, capsules, ovules,elixirs, solutions or suspensions, which may contain flavouring orcolouring agents, for immediate-, delayed- or controlled-releaseapplications. The compounds of the invention may also be administeredvia intracavernosal injection.

Such tablets may contain excipients such as microcrystalline cellulose,lactose, sodium citrate, calcium carbonate, dibasic calcium phosphateand glycine, disintegrants such as starch (preferably corn, potato ortapioca starch), sodium starch glycollate, croscarmellose sodium andcertain complex silicates, and granulation binders such aspolyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC),hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia.Additionally, lubricating agents such as magnesium stearate, stearicacid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers ingelatin capsules. Preferred excipients in this regard include lactose,starch, a cellulose, milk sugar or high molecular weight polyethyleneglycols. For aqueous suspensions and/or elixirs, the compounds of theinvention may be combined with various sweetening or flavouring agents,colouring matter or dyes, with emulsifying and/or suspending agents andwith diluents such as water, ethanol, propylene glycol and glycerin, andcombinations thereof.

The compounds of the invention can also be administered parenterally,for example, intravenously, intra-arterially, intraperitoneally,intrathecally, intraventricularly, intrasternally, intracranially,intra-muscularly or subcutaneously, or they may be administered byinfusion techniques. They are best used in the form of a sterile aqueoussolution which may contain other substances, for example, enough saltsor glucose to make the solution isotonic with blood. The aqueoussolutions should be suitably buffered (preferably to a pH of from 3 to9), if necessary. The preparation of suitable parenteral formulationsunder sterile conditions is readily accomplished by standardpharmaceutical techniques well-known to those skilled in the art.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

For oral and parenteral administration to human patients, the dailydosage level of the compounds of the invention will usually be from 1mg/kg to 30 mg/kg. Thus, for example, the tablets or capsules of thecompound of the invention may contain a dose of active compound foradministration singly or two or more at a time, as appropriate. Thephysician in any event will determine the actual dosage, which will bemost suitable for any individual patient, and it will vary with the age,weight and response of the particular patient. The above dosages areexemplary of the average case. There can, of course, be individualinstances where higher or lower dosage ranges are merited and such arewithin the scope of this invention.

The compounds of the invention can also be administered intranasally orby inhalation and are conveniently delivered in the form of a dry powderinhaler or an aerosol spray presentation from a pressurised container,pump, spray or nebuliser with the use of a suitable propellant, e.g.dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoro-ethane, a hydrofluroalkane such as1,1,1,2-tetrafluoroethane (HFA 134A3 or 1,1,1,2,3,3,3-heptafluoropropane(HFA 227EA3), carbon dioxide or other suitable gas. In the case of apressurised aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. The pressurised container, pump,spray or nebuliser may contain a solution or suspension of the activecompound, e.g. using a mixture of ethanol and the propellant as thesolvent, which may additionally contain a lubricant, e.g. sorbitantrioleate. Capsules and cartridges (made, for example, from gelatin) foruse in an inhaler or insufflator may be formulated to contain a powdermix of a compound of the invention and a suitable powder base such aslactose or starch.

Aerosol or dry powder formulations are preferably arranged so that eachmetered dose or “puff” delivers an appropriate dose of a compound of theinvention for delivery to the patient. It will be appreciated that theoverall daily dose with an aerosol will vary from patient to patient,and may be administered in a single dose or, more usually, in divideddoses throughout the day.

Alternatively, the compounds of the invention can be administered in theform of a suppository or pessary, or they may be applied topically inthe form of a lotion, solution, cream, ointment or dusting powder. Thecompounds of the invention may also be transdermally administered, forexample, by the use of a skin patch. They may also be administered bythe ocular route, particularly for treating diseases of the eye.

For ophthalmic use, the compounds of the invention can be formulated asmicronised suspensions in isotonic, pH adjusted, sterile saline, or,preferably, as solutions in isotonic, pH adjusted, sterile saline,optionally in combination with a preservative such as a benzylalkoniumchloride. Alternatively, they may be formulated in an ointment such aspetrolatum.

For application topically to the skin, the compounds of the inventioncan be formulated as a suitable ointment containing the active compoundsuspended or dissolved in, for example, a mixture with one or more ofthe following: mineral oil, liquid petrolatum, white petrolatum,propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifyingwax and water. Alternatively, they can be formulated as a suitablelotion or cream, suspended or dissolved in, for example, a mixture ofone or more of the following: mineral oil, sorbitan monostearate, apolyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax,cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavoured basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouth-washes comprising the active ingredient in asuitable liquid carrier.

Generally, in humans, oral or topical administration of the compounds ofthe invention is the preferred route, being the most convenient. Incircumstances where the recipient suffers from a swallowing disorder orfrom impairment of drug absorption after oral administration, the drugmay be administered parenterally, e.g. sublingually or buccally.

For veterinary use, a compound of the invention is administered as asuitably acceptable formulation in accordance with normal veterinarypractice and the veterinary surgeon will determine the dosing regimenand route of administration, which will be most appropriate for aparticular animal.

1. An isolated human monoclonal antibody molecule or fragment thereofthat selectively binds to ICAM-1 on the surface of a target cell and, onbinding ICAM-1, induces apoptosis of the target cell, wherein theantibody or fragment has variable regions having the sequences set forthin SEQ ID NOs: 6 and
 8. 2. The isolated antibody molecule of claim 1wherein the target cell is an immune cell or an epithelial cell.
 3. Theisolated antibody molecule of claim 2 wherein the immune cell is a Blymphocyte.
 4. The isolated antibody molecule of claim 1 wherein thetarget cell is associated with a disease.
 5. The isolated antibodymolecule of claim 4 wherein the disease is selected from the groupconsisting of: cancer; autoimmune diseases; an acute inflammatorydisorder; a chronic inflammatory disorder; sepsis and infectiousdisease, wherein said autoimmune diseases are selected from the groupconsisting of rheumatoid arthritis, and Systemic lupus erythematosus(SLE).
 6. The isolated antibody molecule of claim 5 wherein the diseaseis a cancer selected from lymphoma, leukaemia, myeloma, gastric cancer,breast cancer, liver cancer, lung cancer, melanoma, bladder cancer,choroid cancer, pancreatic cancer, colon cancer and prostate cancer. 7.The isolated antibody molecule of claim 1 wherein the antibody moleculeis an IgG.
 8. The antibody molecule of claim 7 wherein the antibody is asingle chain antibody selected from the group of an IgG₁, IgG₂, IgG₃ orIgG₄.
 9. A pharmaceutical composition comprising a human monoclonalantibody molecule or a fragment thereof, and apharmaceutically-acceptable carrier, excipient or diluent, wherein saidantibody molecule or fragment selectively binds to ICAM-1 on the surfaceof a target cell, and on binding ICAM-1, induces apoptosis of the targetcell, wherein the antibody or fragment has variable regions having thesequences set forth in SEQ ID NOs: 6 and
 8. 10. The isolated antibody ofclaim 5, wherein said infectious disease is HIV.
 11. A method ofinducing apoptosis in a target cell comprising the steps: a. providingone or more target cells displaying the cell surface antigen, ICAM-1; b.providing a human monoclonal antibody molecule or fragment thereof whichselectively binds to cell surface ICAM-1 and, on binding ICAM-1, inducesapoptosis of the target cell, wherein the antibody or fragment hasvariable regions having the sequences set forth in SEQ ID NOs: 6 and 8;and c. exposing the target cells of (a) to the antibody molecule orfragment of (b) to induce apoptosis in the target cells.
 12. An in vitromethod of inducing apoptosis in a target cell comprising the steps of:a. providing one or more target cells; b. providing a human monoclonalantibody molecule or fragment thereof that selectively bind to cellsurface ICAM-1 of said target cells, wherein the antibody or fragmenthas variable regions having the sequences set forth in SEQ ID NOs: 6 and8; and c. exposing the target cells of (a) to the antibody molecule orfragment of (b) so as to induce apoptosis in the target cells.
 13. An invitro method as claimed in claim 12 wherein the target cells provided instep (a) are immune cells or epithelial cells.
 14. An in vitro method asclaimed in claim 13 wherein the immune cells are B-lymphocytes.
 15. Anin vitro method as claimed in claim 14 wherein the disease is a cancerselected from lymphoma, leukaemia, myeloma gastric cancer, breastcancer, liver cancer, lung cancer, melanoma, bladder cancer, choroidcancer, colon cancer, and prostate cancer.
 16. An in vivo method ofinducing apoptosis in a target cell comprising the steps of: a.providing one or more target cells; b. providing one or more humanmonoclonal antibody molecules or fragments thereof that selectively bindto cell surface ICAM-1 of said target cells, wherein the antibody orfragment has variable regions having the sequences set forth in SEQ IDNOs: 6 and 8; and c. exposing the target cells of (a) to the antibodymolecules or fragments of (b) so as to induce apoptosis in the targetcells.
 17. An in vivo method as claimed in claim 16 wherein the targetcells provided in step (a) are immune cells or epithelial cells.
 18. Anin vivo method as claimed in claim 17 wherein the immune cells areB-lymphocytes.
 19. An in vivo method as claimed in claim 16 wherein thedisease is a cancer selected from lymphoma, leukaemia, myeloma, gastriccancer, breast cancer, liver cancer, lung cancer, melanoma, bladdercancer, choroid cancer, pancreatic cancer, colon cancer and prostatecancer.